Objective: To describe the submucosal microbial profiles of peri-implantitis and healthy implants, and to explore bacteria that might be correlated with clinical parameters.
Methods: In the present cross-sectional study, 49 patients were recruited. Each patient contributed with one implant, submucosal biofilms were collected from 20 healthy implants and 29 implants with peri-implantitis. DNA was extracted and bacterial 16S ribosomal RNA (16S rRNA) genes were amplified. Submucosal biofilms were analyzed using 16S rRNA sequencing at Illumina MiSeq platform. Differences between the groups were determined by analyzing α diversity, microbial component and microbial structure. The potential correlation between the bacteria with pocket probing depth (PPD) of peri-implant calculated by Spearman correlation analysis.
Results: The α diversity of submucosal microbial of health group was significantly lower than that in peri-implantitis group (Chao1 index: 236.85±66.13 vs. 150.54±57.43, P < 0.001; Shannon index: 3.42±0.48 vs. 3.02±0.65, P=0.032). Principal coordinated analysis showed that the submucosal microbial structure had significant difference between healthy and peri-implantitis groups [R2=0.243, P=0.001, analysis of similarities (ANOSIM)]. Compared with healthy implants, relative abundance of periodontal pathogens were higher in peri-implantitis, including members of the red complex (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola) and some members of orange complex (Precotella intermedia, Eubacterium nodatum, Parvimonas micra), as well as some new periodontal pathogens, such as Fillifactor alocis, Fretibacterium fastidiosum, Desulfobulbus sp._HMT_041, and Porphyromonas endodontalis. Spearman correlation analysis revealed that the relative abundance of Treponema denticola (r=0.686, P < 0.001), Tannerella forsythia (r=0.675, P < 0.001), Fretibacterium sp. (r=0.671, P < 0.001), Desulfobulbus sp._HMT_041 (r=0.664, P < 0.001), Filifactor alocis (r=0.642, P < 0.001), Fretibacterium fastidiosum (r=0.604, P < 0.001), Porphyromonas gingivalis (r=0.597, P < 0.001), Porphyromonas endodontalis (r=0.573, P < 0.001) were positive correlated with PPD. While the relative abundance of Rothia aeria (r=-0.615, P < 0.001) showed negatively correlation with PPD.
Conclusion: Marked differences were observed in the microbial profiles of healthy implants and peri-implantitis. The members of red and orange complex as well as some new periodontal pathogens seem to play an important role in peri-implant disease. Compared with healthy implants, the submucosal microbial of peri-implantitis were characterized by high species richness and diversity.
目的: 探索种植体黏膜下微生物在健康种植体和种植体周炎中的构成与差异，并分析与临床指标存在相关性的菌种，为种植体周炎的病因学研究提供参考。
方法: 采用横断面研究，共纳入49例患者，20例为健康种植体，29例为种植体周炎，共采集49份黏膜下微生物样本进行16S核糖体RNA(16S ribosomal RNA, 16S rRNA)基因高通量测序。对两组样本的多样性、菌群构成和差异物种进行分析和比较，采用Spearman相关性分析评价菌种与探诊深度(probing pocket depth, PPD)之间的相关性。
结果: 健康组的α多样性显著低于种植体周炎组[Chao1指数：236.85±66.13 vs. 150.54±57.43，P < 0.001；Shannon指数：3.42±0.48 vs. 3.02±0.65，P=0.032]。主成分分析显示，两组样本的群落结构差异有统计学意义[相似性分析(analysis of similarities, ANOSIM)，R2=0.243，P=0.001]。与健康种植体相比，种植体周炎黏膜下菌斑中的牙周致病菌显著增加，包括红色复合体成员[牙龈卟啉单胞菌(Porphyromonas gingivalis)、福赛斯坦纳菌(Tannerella forsythia)、齿垢密螺旋体(Treponema denticola)]、部分橙色复合体成员[中间普氏菌(Precotella intermedia)、缠结优杆菌(Eubacterium nodatum)和微小微单胞菌(Parvimonas micra)]以及一些新型牙周致病菌，如龈沟产线菌(Fillifactor alocis)、苛求依赖杆菌(Fretibacterium fastidiosum)、牙髓卟啉单胞菌(Porphyromonas endodontalis)和Desulfobulbus sp._HMT_041。相关性分析显示，种植体黏膜下微生物中，齿垢密螺旋体(r=0.686, P < 0.001)、福赛斯坦纳菌(r=0.675, P < 0.001)、Fretibacterium sp. (r=0.671, P < 0.001)、Desulfobulbus sp._HMT_041 (r=0.664, P < 0.001)、龈沟产线菌(r=0.642, P < 0.001)、苛求依赖杆菌(r=0.604, P < 0.001)、牙龈卟啉单胞菌(r=0.597, P < 0.001)、牙髓卟啉单胞菌(r=0.573, P < 0.001)的丰度与种植体周PPD呈显著正向相关；空间罗氏菌(Rothia aeria)(r=-0.615, P < 0.001)的丰度与PPD呈显著负相关。
结论: 种植体黏膜下微生物在健康种植体和种植体周炎中的群落构成存在明显不同，红色复合体、部分橙色复合体成员以及一些新型牙周致病菌与种植体周炎的发生密切相关；与健康种植体相比，种植体周炎的黏膜下微生物群落具有高物种丰富度及多样性的特点。
Keywords: Dental implants; Human microbiota; Illumina sequencing; Peri-implantitis.